OpenFIBSEM: A universal API for FIBSEM control.

This paper introduces OpenFIBSEM, a universal API to control Focused Ion Beam Scanning Electron Microscopes (FIBSEM). OpenFIBSEM aims to improve the programmability and automation of electron microscopy workflows in structural biology research. The API is designed to be cross-platform, composable, and extendable: allowing users to use any portion of OpenFIBSEM to develop or integrate with other software tools. The package provides core functionality such as imaging, movement, milling, and manipulator control, as well as system calibration, alignment, and image analysis modules. Further, a library of reusable user interface components integrated with napari is provided, ensuring easy and efficient application development. OpenFIBSEM currently supports ThermoFisher and TESCAN hardware, with support for other manufacturers planned. To demonstrate the improved automation capabilities enabled by OpenFIBSEM, several example applications that are compatible with multiple hardware manufacturers are discussed. We argue that OpenFIBSEM provides the foundation for a cross-platform operating system and development ecosystem for FIBSEM systems. The API and applications are open-source and available on GitHub (https://github.com/DeMarcoLab/fibsem).

Three-dimensional imaging on a chip using optofluidics light-sheet fluorescence microscopy.

Volumetric, sub-micron to micron level resolution imaging is necessary to assay phenotypes or characteristics at the sub-cellular/organelle scale. However, three-dimensional fluorescence imaging of cells is typically low throughput or compromises on the achievable resolution in space and time. Here, we capitalise on the flow control capabilities of microfluidics and combine it with microoptics to integrate light-sheet based imaging directly into a microfluidic chip. Our optofluidic system flows suspended cells through a sub-micrometer thick light-sheet formed using micro-optical components that are cast directly in polydimethylsiloxane (PDMS). This design ensures accurate alignment, drift-free operation, and easy integration with conventional microfluidics, while providing sufficient spatial resolution, optical sectioning and volumetric data acquisition. We demonstrate imaging rates of 120 ms per cell at sub-µm resolution, that allow extraction of complex cellular phenotypes, exemplified by imaging of cell clusters, receptor distribution, and the analysis of endosomal size changes.

Hybrid refractive-diffractive axicons for Bessel-beam multiplexing and resolution improvement.

Optical elements rely on refraction, diffraction, or reflection for light manipulation. Fusing diffractive and refractive functions in a single element provides an extra layer of control over the wave propagation, allowing complex beam shaping through self-aligned, monolithic and miniaturized optics. Using gray-scale lithography with high-current focused Xe ion-beams, we realized hybrid refractive-diffractive micro-axicons that feature diffractive gratings engraved on their conical surfaces. Furthermore, we fabricated these devices in lithium niobate, which is a challenging piezo/optoelectronic material for processing with an as-yet unexploited potential in optical applications. The curvilinear surfaces of fabricated micro-axicons with a 230-µm diameter were engraved with diffraction linear and circular gratings of various depths (<400 nm), and the optical performance of these components was characterized, showing excellent agreement with theoretical expectations. The fusing of diffractive elements with carrier refractive surfaces introduces additional or enhanced device functionalities, such as beam multiplexing and resolution improvement. The potential applications of such monolithic and miniaturized hybrid micro-optical components include beamshaping for fluorescence microscopy.

Assembly and Imaging set up of PIE-Scope.

Cryo-Electron Tomography (cryo-ET) is a method that enables resolving the structure of macromolecular complexes directly in the cellular environment. However, sample preparation for in situ Cryo-ET is labour-intensive and can require both cryo-lamella preparation through cryo-Focused Ion Beam (FIB) milling and correlative light microscopy to ensure that the event of interest is present in the lamella. Here, we present an integrated cryo-FIB and light microscope setup called the Photon Ion Electron microscope (PIE-scope) that enables direct and rapid isolation of cellular regions containing protein complexes of interest. The PIE-scope can be retrofitted on existing microscopes, although the drawings we provide are meant to work on ThermoFisher DualBeams with small mechanical modifications those can be adapted on other brands.

PIE-scope, integrated cryo-correlative light and FIB/SEM microscopy.

Cryo-electron tomography (cryo-ET) is emerging as a revolutionary method for resolving the structure of macromolecular complexes in situ. However, sample preparation for in situ Cryo-ET is labour-intensive and can require both cryo-lamella preparation through cryo-focused ion beam (FIB) milling and correlative light microscopy to ensure that the event of interest is present in the lamella. Here, we present an integrated cryo-FIB and light microscope setup called the Photon Ion Electron microscope (PIE-scope) that enables direct and rapid isolation of cellular regions containing protein complexes of interest. Specifically, we demonstrate the versatility of PIE-scope by preparing targeted cryo-lamellae from subcellular compartments of neurons from transgenic Caenorhabditis elegans and Drosophila melanogaster expressing fluorescent proteins. We designed PIE-scope to enable retrofitting of existing microscopes, which will increase the throughput and accuracy on projects requiring correlative microscopy to target protein complexes. This new approach will make cryo-correlative workflow safer and more accessible.

Fabrication of glass microlenses using focused Xe beam.

Focused ion beam (FIB) systems based on high brightness plasma ion sources are becoming largely diffuse in material and semiconductor research, thanks to the higher current densities and milling rates provided by noble gas ions (e.g., Xe) compared with traditional liquid metal Ga FIBs. In this paper, we demonstrate the feasibility of a rapid, direct milling of microlenses in glass substrates using high current Xe plasma FIB. We present quantitative analyses of roughness and profile of microlenses with diameters up to 230-µm and focal distances between 7 mm and 1.4 mm. We characterized the performance of the lenses by mapping the transmitted intensity through the lenses, by forming an image of a resolution object by scanning the focused spot and collecting the transmitted intensity, and in full-field imaging experiments. The results indicate the applicability of plasma focused ion beam systems for direct writing in glass of high-quality micro-optical elements with diffraction-limited focusing.

Refractive micro-lenses and micro-axicons in single-crystal lithium niobate.

The high refractive index of lithium niobate crystal (n = 2.2) and the highly transparent range (300-5000 nm), makes it a perfect material for refractive lenses and other types of micro-optical elements. This material already finds extensive use in waveguides and photonic crystals, however, little work has been done on producing refractive optical components in lithium niobate, presumably due to the challenges associated with its fragility and difficulties in three-dimensional micromachining. In this study, we fabricated high-quality refractive micro-lenses and micro-axicons with low surface roughness (< λvis / 20), with 220 µm diameters and sag heights up to 22 µm in single-crystal LN using focused Xe beam milling. Xe ion beam milling is a flexible and rapid technique allowing realization of complex three-dimensional surface reliefs directly in lithium niobate. We characterized the optical performance of the fabricated elements showing sub-µm focusing capabilities of both the lenses and axicons.

High aspect ratio plasmonic nanostructures for sensing applications.

We present an experimental and theoretical study of plasmonic modes in high aspect ratio nanostructures in the visible wavelength region and demonstrate their high performance for sensing applications. Ordered and well-defined plasmonic structures with various cross-sectional profiles and heights are obtained using a top-down fabrication process. We show that, compared to cylindrical nanorods, structures with split-ring resonator-like cross sections have great potential for powerful sensing due to a pronounced polarization dependence, strong field enhancement, structural tunability, and improved mechanical stability. The plasmonic structures under study exhibit high sensitivities, up to nearly 600 nm/RIU, and figures of merit above 20.

High-efficiency Fresnel zone plates for hard X-rays by 100 keV e-beam lithography and electroplating.

The fabrication and characterization of Fresnel zone plates (FZPs) for hard X-ray microscopy applications are reported. High-quality 500 nm- and 1 µm-thick Au FZPs with outermost zone widths down to 50 nm and 70 nm, respectively, and with diameters up to 600 µm were fabricated. The diffraction efficiencies of the fabricated FZPs were measured for a wide range of X-ray energies (2.8-13.2 keV) showing excellent values up to 65-75% of the theoretical values, reflecting the good quality of the FZPs. Spatially resolved diffraction efficiency measurements indicate the uniformity of the FZPs and a defect-free structure.

Ultra-high resolution zone-doubled diffractive X-ray optics for the multi-keV regime.

X-ray microscopy based on Fresnel zone plates is a powerful technique for sub-100 nm resolution imaging of biological and inorganic materials. Here, we report on the modeling, fabrication and characterization of zone-doubled Fresnel zone plates for the multi-keV regime (4-12 keV). We demonstrate unprecedented spatial resolution by resolving 15 nm lines and spaces in scanning transmission X-ray microscopy, and focusing diffraction efficiencies of 7.5% at 6.2 keV photon energy. These developments represent a significant step towards 10 nm spatial resolution for hard X-ray energies of up to 12 keV.

Direct e-beam writing of dense and high aspect ratio nanostructures in thick layers of PMMA for electroplating.

Due to the ability of 100 keV electrons to penetrate deep into resist with little scattering, we were able to directly write various dense and high aspect ratio nanostructures in 540 nm and 1.1 microm thick layers of poly(methyl methacrylate) (PMMA) resist. The PMMA molds produced by electron beam lithography were developed using a high contrast developer. The molds were used to transfer the pattern into metallic nanostructures by filling the developed trenches with Au by electroplating. By exposing lines narrower than the target width, we observed improved process latitude and line width control. The obtained aspect ratios of the dense structures are nearly 20 in 1.1 microm PMMA layers and > 16 for structures electroplated into this PMMA mold. The fabrication method was successfully applied to produce Au diffractive x-ray Fresnel zone plates of exceptionally good quality with 50 and 70 nm outermost zones using 540 nm and 1.1 microm thick PMMA molds. In addition, we also produced regular arrays of high aspect ratio and dense Au nanorods with periods down to 100 nm and high aspect ratio split-ring resonators.

Dense high aspect ratio hydrogen silsesquioxane nanostructures by 100 keV electron beam lithography.

We investigated the fabrication of dense, high aspect ratio hydrogen silsesquioxane (HSQ) nanostructures by 100 keV electron beam lithography. The samples were developed using a high contrast developer and supercritically dried in carbon dioxide. Dense gratings with line widths down to 25 nm were patterned in 500 nm-thick resist layers and semi-dense gratings with line widths down to 10 nm (40 nm pitch) were patterned in 250 nm-thick resist layers. The dense HSQ nanostructures were used as molds for gold electrodeposition, and the semi-dense HSQ gratings were iridium-coated by atomic layer deposition. We used these methods to produce Fresnel zone plates with extreme aspect ratio for scanning transmission x-ray microscopy that showed excellent performance at 1.0 keV photon energy.

Mobility determination of lead isotopes in glass for retrospective radon measurements.

In retrospective radon measurements, the 22-y half life of (210)Pb is used as an advantage. (210)Pb is often considered to be relatively immobile in glass after alpha recoil implanted by (222)Rn progenies. The diffusion of (210)Pb could, however, lead to uncertain wrong retrospective radon exposure estimations if (210)Pb is mobile and can escape from glass, or lost as a result of cleaning-induced surface modification. This diffusion was studied by a radiotracer technique, where (209)Pb was used as a tracer in a glass matrix for which the elemental composition is known. Using the ion guide isotope separator on-line technique, the (209)Pb atoms were implanted into the glass with an energy of 39 keV. The diffusion profiles and the diffusion coefficients were determined after annealing at 470-620 degrees C and serial sectioning by ion sputtering. In addition, the effect of surface cleaning on diffusion was tested. From the Arrhenius fit, the activation enthalpy (H) was determined, which is equal to 3.2 +/- 0.2 eV, and also the pre-exponential factor D(0), in the order of 20 m(2)s(-1). This result confirms the assumption that over a time period of 50 y (209)Pb (and (210)Pb) is effectively immobile in the glass. The boundary condition obtained from the measurements had the characteristic of a sink, implying loss of (209)Pb in the topmost surface at high temperatures.